Half of each PCR products were electrophoresed on 1% agarose gels, and their amounts were evaluated by staining with ethidium bromide.

DH-JH recombination was detected as amplified fragments of 1,033 bp, 716 bp and 333 bp using a primer DHL(5’) and J3(3’). Germline alleles were detected as an amplified fragment of 1,259 bp using a primer Mu0(5’) and J3(3’).

Results

Figure 1.DNA PCR assays of germline (GL) or DH-JH rearranged Igh chain (DJ) genes were performed with mouse splenocytes. A PCR experiment using a primer DHL(5’) and J3(3’) can detect three types of DH-JH rearrangement (J1, J2, and J3) (Schlissel et al., 1991). All of three bands are present with successful DH-JH rearrangement. However, a J1 band is sometimes undetected in the ethidium bromide-based DNA-band visualization when the amount of template DNA is very small. The size marker was loaded in the left lane.

Acknowledgments

This protocol was adapted from a previously published paper by Schlissel et al. (1991). The representative data shown in the protocol was adapted from Satoh et al. (2013).